Willy
Burgdorfer, PhD, MD (Hon)
Rocky Mountain Laboratories
National Institutes of Health, National Institute of Allergy
and Infectious Diseases
Increase
Evidence Of Mosquito / Spirochete Associations
The
recent demonstration and isolation of spirochetes (including
the Lyme disease agent, Borrelia afzelii) in and from
Aedes and Culex mosquitoes in southern Moravia
(Czech Republic) promoted this short review of literature pertaining
to the association of spirochetes with mosquitoes.
Reports
as early as 1904 describe the presence of spirochetes in intestinal
tracts of larval mosquitoes as well as in malpighian tubules
and salivary glands of adult Culex, Anopheles and Aedes
mosquitoes. Yet, there is no information as to the ability of
these insects to transmit spirochetes by bite. An exception is
a report on the successful transmission of Borrelia anserina
by experimentally infected Aedes aegypti. Similar experimental
infection and transmission studies of the Lyme disease agent
Borrelia burgdorferi sensu stricto in three species of
mosquitoes (Ae. aegypti, Ae. atropalpus, Ae. triseriatus)
showed the duration of spirochetal infections in the intestinal
tracts of these insects to be ephemeral and not involving salivary
gland tissues. In contrast are the recent reports from southern
Moravia where over-wintering Aedes and Culex have
been found naturally infected with spirochetes. One strain isolated
from Ae. vexans was identified as B. afzelii, two
other strains from C. pipiens molestus appeared to be
new hitherto undescribed spirochetes. Studies are in progress
to determine whether these spirochetes produce in their mosquito
vectors systemic infections including the tissues of salivary
glands from where they could be transmitted via saliva during
feeding on animal hosts and possibly humans.
Claire
M. Fraser, PhD
Vice-President for Research, The Institute for Genomic Research
Complete
Genome Sequence of Borrelia burgdorferi
Spirochetes
from the genus Borrelia were found to be the etiologic
agent of Lyme disease in the United States in 1982. Lyme disease
is a multi-systemic disease, which can become chronic if left
untreated, and its causative agent, B. burgdorferi, is
carried by ticks, mostly in the genus Ixodes. Lyme disease
is currently the most prevalent tick-borne disease in the United
States. The type strain, B31 was isolated from an Ixodes scapularis
tick on Shelter Island, New York. We report here the complete
genome sequencing of B. burgdorferi isolate B31. The genome
contains a linear chromosome of 910,725 bp and 21 linear and
circular plasmids with a combined size of more than 600,000 bp.
The chromosome contains 853 genes encoding a basic set of proteins
for DNA replication, transcription, translation, solute transport,
and energy metabolism, but no genes for cellular biosynthetic
reactions, similar to Mycoplasma genitalium. Since B.
burgdorferi and M. genitalium are distantly related
eubacteria, we suggest that their limited metabolic capacities
reflect convergent evolution by gene loss from a more metabolically
competent progenitor. Eight hundred thirty eight genes were identified
on 21 plasmids, the majority of which have no known biological
function. Seventy percent of plasmid genes are paralogs that
comprise 47 gene families; a large number of these genes encode
putative lipoproteins. The biological significance of the multiple
plasmid-encoded genes is not clear, although they may play a
role in antigenic variation or immune evasion.
Jon
T. Skare, PhD
Dept. of Medical Microbiology & Immunology, Texas A&
M University, Health Science Center
Identification
and Characterization of Virulent Strain Associated Outer Membrane
Proteins of Bb
Virulent
strain associated (VSA) outer membrane proteins of Borrelia
burgdorferi mediate the initial borrelial-host interaction
and, as such, are candidate virulence determinants. To identify
such proteins, we used serum from rabbits immune to re-infection
with virulent B. burgdorferi sensu stricto strain B31
adsorbed against avirulent B. burgdorferi to obtain a
reagent consisting of antibodies specific for VSA antigens. This
VSA-specific serum was then used to screen immunoreactive plaques
from a B. burgdorferi expression library. Sequence analysis
indicated that the 16 immunoreactive B. burgdorferi antigens
were encoded by 9 genes, including the locus required for decorin
binding (dbpAB) and an additional lipoprotein antigen
containing 22 consecutive 9 amino acid repeats that we have designated
vraA for VSA repetitive antigen A.
Of the 9 genes identified in this screen, 8 were encoded on plasmids
known to be lost during in vitro cultivation with a concomitant
loss of infectivity in animal models of Lyme borreliosis. We
have subsequently determined that DbpA, DbpB and VraA are derepressed
at 37°C relative to cells grown at either 23°C or 32°C,
suggesting that they are expressed at high levels within mammalian
hosts.
These
results, along with other previous observations, indicate that
B. burgdorferi alters its protein and antigenic profile
within the mammalian host(s) relative to cells cultivated in
vitro. As such, the identification of antigens expressed
at higher levels within infected mammals, such as DbpA, DbpB,
and VraA, provide additional logical vaccine candidates to complement
the current OspA vaccine regimen. Finally, with the advent of
the B. burgdorferi genome project, it should be possible
to assess the global regulation of genes encoding VSA antigens
to determine the environmental cues that regulate B. burgdorferi
VSA gene expression.
Maria
M. Picken, MD, PhD
Associate Professor of Pathology, Loyola University Medical Center
Survey
for Borrelia Species Among Reservoir Animals Captured
in Forested Areas of Greater Metropolitan Chicago
Picken MM*1, Cera LM1, Drummond F2,
Elamma CA1, Anchor C3, and Picken RN4
Loyola University Medical Center (1), Maywood, IL; Lake County
Forest Preserve (2), Libertyville, IL; Cook County Forest Preserve
(3), River Forest, IL; Hines VA Hospital (4), Maywood, IL
Objectives:
To survey the small animal reservoir of forested areas in northwestern
and north suburban Chicago for the presence of B. burgdorferi,
to isolate spirochetes in culture, and to characterize the resulting
isolates by molecular genotypic analysis.
Methods:
During summer 1996, 126 small animals were trapped in northwestern
Cook Co. and cultured for B. burgdorferi. These comprised
106 Peromyscus leucopus, 8 Tamias striatus, 5 Microtus
pennsylvanicus, 3 Spermophilus tridecemlineatus, 1
Marmota monax, 1 Peromyscus maniculatus, 1 Sorex
cinereus, and 1 Zapus hudsonius. During summer 1997,
56 animals were trapped in Lake County, north of Chicago, and
cultured. These comprised 16 P. leucopus, 34 M. pennsylvanicus,
3 P. maniculatus , and 3 Z. hudsonius . Borrelia
isolates were characterized by pulsed-field gel electrophoretic
(PFGE) analysis of their plasmid content, and chromosomal macrorestriction
patterns as well as nucleotide sequence determination of the
rrf (5S)-rrl (23S) intergenic spacer region.
Results:
Two isolates were obtained from Cook Co. in 1996, from P.
leucopus and M. pennsylvanicus. In 1997, 8 isolates
were obtained form 5 animals captured in Lake Co., comprising
4 M. pennsylvanicus and 1 Z. hudsonius. In the
case of 3 M. pennsylvanicus, isolates were obtained from
both urinary bladder and ear snips. PFGE analysis of MluI-digested
genomic DNA from the isolates demonstrated two patterns of high
molecular mass fragments identical to those of B. burgdorferi
strains DN127 (2 Cook Co. isolates) and 25015 (8 Lake Co. isolates).
Plasmid patterns and low molecular mass fragments showed differences
between the 10 strains. Sequence analysis of the rrf-rrl
intergenic spacer region demonstrated closely similar sequences
to those reported for DN127 and 25015, but also showed individual
nucleotide differences between strains.
Conclusions:
These studies demonstrate the presence of DN127-group B. burgdorferi
infecting the rodent population in northwestern and north suburban
forest areas of Chicago. The prevalence of these organisms is
clearly higher than was previously supposed. Whether DN127-group
borrelia represent a human Lyme disease risk has yet to be determined.
Mark
J. Cartwright, PhD Candidate, Boston University Medical Center
Suzanne E. Martin, Research Assistant, Boston University Medical
Center
Toxins
of Borrelia burgdorferi
Mark J. Cartwright*, Suzanne E. Martin, and Sam T. Donta, Boston
University Medical Center
The
mechanisms responsible for many of the symptoms in patients with
Lyme disease remain to be delineated. The organism can only rarely
be found following the initial infection and dissemination, suggesting
that for persistent infection the reservoir is intracellular.
Because so many of the symptoms appear to be related to the nervous
system, it is postulated that the Lyme spirochetes reside in
neurons themselves, glial cells, or endothelial cells that provide
the nervous system with its blood supply. It is further postulated
that toxins are released by the borrelial organisms that then
interfere with normal neurochemical function.
The
purpose of this research program is to identify toxins of B.
burgdorferi that may directly or indirectly impact on normal
neurophysiology. Towards that goal, we designed "degenerate"
primers to highly conserved regions within toxin sequences. We
used these primers for PCR to identify genes that express proteins
analogous to existing toxins. To date, we have identified and
cloned B. burgdorferi genes derived from cholera, diphtheria,
and pertussis toxins. The putative toxin genes are identical
to several unidentified genes contained in the recently published
complete DNA sequence of B. burgdorferi. In addition,
one of the putative toxin genes is conserved to a Treponema
pallidum gene of undefined function. As a second approach
to identify possible toxins, assays (e.g. ribosylation) were
performed on both the B. burgdorderi bacteria and its
conditioned media, in which enzymatic activity was detected.
David
W. Dorward, PhD
Microbiologist, National Institutes of Health, Rocky Mountain
Laboratories
In
Vitro
Evidence for Lymphocytic Membrane Cloaking by Borrelia burgdorferi
David W. Dorward* and Elizabeth R. Fischer. NIH/Rocky Mountain
Labs
In
vitro
studies have demonstrated that Lyme disease spirochetes including
Borrelia burgdorferi and B. garinii attach to,
invade, and kill subsets of human B and T lymphocytes. To further
understand such interactions, temperature-regulated co-incubation
mixtures were examined by immunofluorescence and electron microscopy.
Low passage (<8) B. burgdoferi Sh-2-82 and SKW 6.4
B cells were mixed at 10:1 ratios at 4°C for 1 hr, then warmed
to 37°C and followed. Spirochetes attached to nearly 50%
of B cells in all mixtures. Whereas, attachment peaked after
1 hr at 37°, invasion appeared to peak at 4 hrs. Addition
of 0.1-1% carboxymethycellulose dramatically enhanced the rate
of spirochetal motility, but did not increase cell invasion.
No evidence for degradation of nor damage to intracellular spirochetes
was detected or observed. Emergence form B cells was either lytic
or non-lytic. Emergent spirochetes frequently retained lymphocytic
membrane envelopes. Video microscopy revealed that enveloped
spirochetes had normal motility. After 24 hrs one third of co-incubated
spirochetes labeled with antibodies to human B cell antigens.
Relatively few spirochetes remained enveloped after 48hrs. Immune
electron microscopy showed that although such enveloped spirochetes
exhibited surface-exposed B cell antigens, anti-OspA antibodies
failed to bind. Furthermore, spirochetes incubated with B cells
acquired a time-dependent resistance to classic complement-mediated
killing. Such results suggest that in vitro interactions
with cultured human B cells result in B. burgdorferi retaining
one or more layers of lymphocytic membrane that mask spirochetal
antigens, and possible interfere with antibody-mediated recognition
and neutralization. If such interactions occur in vivo,
such a process could represent a previously unrecognized bacterial
virulence strategy.
John
Anderson, PhD
Director, Connecticut Agricultural Experiment Station
Tick
Vectors of Borrelia burgdorferi
There
are about 839 different species of ticks in the world, of which
almost 80 percent are hard bodied ticks. A few of these, namely
Ixodes scapularis and Ixodes pacificus of North
America, and Ixodes ricinus and Ixodes persulcatus,
and possibly Ixodes ovatus in the Old World, are the principal
vectors of spirochetes that cause Lyme disease. These species
have similar natural histories, feed on a wide variety of animals
including mammals, birds and lizards, and all readily feed on
humans. Reservoir hosts of borreliae are primarily rodents. Birds
also harbor borreliae and can transport these pathogens and tick
vectors relatively long distances during migration. Ixodes
scapularis remains abundant in eastern and mid-western United
States because of the ever increasing number of white tail deer.
Personal protection measures are important to reducing exposure
to borreliae.
Edward
Bosler, PhD
SUNY at Stony Brook School of Medicine, Health Science Center
Co-infection
of Mammals and Ticks with Emerging Tick-borne Pathogens
Over
the past several years emerging and re-emerging tick-borne infections
are being diagnosed in humans with greater frequency and may
represent a serious public health problem. In the Northeast and
Midwest the agents of babesiosis (Babesia microti) and
human granulocytic ehrlichiosis (Ehrlichia phagocytophila)
are transmitted by the same vector tick, Ixodes scapularis,
as the Lyme disease bacterium. Evidence from human patients suggests
that co-infection with these causal organisms occurs frequently
and empirically that infection with one or more organisms may
exacerbate or alter the symptoms of another.
During
1997 we assessed the presence of co-infection with these organisms
in potential rodent reservoir hosts, ticks feeding on these hosts
and in free ranging ticks collected from selected areas of Long
Island. Free ranging adult ticks were also examined from areas
of Connecticut and from along the eastern seaboard from Massachusetts
to South Carolina. \Pathogens were directly detected by in
vitro cultivation and indirectly by PCR. PCR proved more
practical for analyzing large numbers of field derived samples.
The rates of co-infection in both hosts and ticks indicate widespread
distribution of the pathogens and that the zoonotic cycles of
each are established on Long Island. These data also indicate
a strong need to establish laboratory "zoonotic" models
for co-infection.
Ibulaimu
Kakoma, DVM, PhD
University of Illinois, College of Veterinary Medicine
Clinical
and Laboratory Characterization of the Canine Monocytic Ehrlichiosis
Syndrome
Ibulaimu Kakoma, D.V.M., Ph.D.*, Richard Hansen, BS* and Jane
Biggerstaff, D.V.M., MS** *College of Veterinary Medicine, Univ.
of Illinois at Urbana-Champaign and ** Jane's Veterinary Clinic,
Texas
When
first described six decades ago in Africa, canine ehrlichiosis
was a mild disease of dogs associated with tick bites. The diagnosis
was clinical and parasitological and the condition was relatively
self-limiting. Subsequently, the classical disease evolved into
a fulminating infection typified by fever, pancytopenia (with
a predominance of thrombocytopenia), a hemorragic crisis which
may include epistaxis, naso-ocular discharge, polyarthritis and
a host of multisystemic metabolic and pathologic disturbances.
The latter may include cardiovascular and CNS complications,
thus making CME, an imitator, next only to Lyme disease.
A breed predisposition has been postulated. The distribution
of cases co-incides with the presence of tick vectors although
mechanical transmission cannot be precluded. These findings prompted
specialists in human infectious diseases to search for a similar
human syndrome.
With
improved technology Ehrlichia canis, the prototype etiologic
agent was cultured and isolated and better diagnostic techniques
(e.g. The Indirect Immunofluorescence, Polymerase Chain Reaction)
have been devised. In addition, clinicians and the lay public
have become more cognizant of potential cases of the disease
and seek to differentiate it from syndromes such as RMSF, Lyme
Disease, Babesiosis and a wide array of autoimmune diseases.
The diagnosis has been complicated by emergence of atypical ehrlichiosis
putatively attributed to different strains of E. canis
and/or E. risticii. Definitive diagnosis is based on a
combination of laboratory findings and a mosaic of clinical observations
consistent with ehrlichiosis. The treatment of choice is tetracycline
and the ideal method of control is elimination of ticks and other
biting vectors. Laboratory studies have been constrained by reliance
on canine models in the absence of a readily available good murine
model.
Profiles
of the syndrome derived from extensive laboratory experimentation
and anecdotal clinical observations highlight the rapidly evolving
nature of this re-emerging syndrome.
Sandra
L. Bushmich, MS, DVM
Associate Professor of Pathobiology, University of Connecticut
Lyme
Disease in Dairy Cattle
In
this talk we will summarize findings on bovine Lyme borreliosis
gleaned from several studies, some of which are in progress.
Lyme disease has been reported in dairy cattle (Post et al. 1986,
Wells et. al. 1993, Burgess et al. 1986, Bushmich 1992). The
most prevalent clinical sign is lameness; erythematous skin rash
has also been described. Serologic diagnosis is hampered by cross
reactivity with other flagellated flora, as well as a high level
of subclinical infection. Our laboratory has conducted several
studies to help define this disease in cattle. Our initial study
involved experimental infection of neonatal calves with Borrelia
burgdorferi (Bb) culture. Infected calves developed a positive
serological response to Bb erythematous skin rash at the
injection site from which Bb were cultured, and shed live
Bb in the urine. Aside from the skin rash, they were clinically
normal. Bb were detected (by culture and/or PCR) in urine
of all 4 infected calves, as well as synovial fluid from one
calf and blood from another. Necropsy cultures from infected
calves were positive for Bb in spleen and synovial tissue
of one calf, and kidney and bladder of another. Control calves
were negative serologically and by culture/PCR. A later detailed
case study involves a mature Holstein cow with initial clinical
sign of severe lameness. Western blot demonstrated Bb
specific antibodies, and skin biopsy was Bb culture and
PCR positive. Physical examination revealed no other cause of
lameness. The cow responded well to a short course of oxytetracycline
treatment, then became lame again. This cow was then moved to
a research facility and treated with alternating penicillin and
oxytetracycline for over 50 days. Although she improved clinically
and returned to the herd, she became severely lame again 2 months
later and was euthanised. Bb was found in synovial tissue,
lymph node, bladder and uterus at necropsy. Studies of natural
Bb infection in bred Holstein heifers are presented as
a separate poster presentation. Preliminary results of experimental
infection of bred dairy heifers with Bb infected and non-infected
control Ixodes scapularis ticks will also be presented.
Reinhard
K. Straubinger, DVM
James A. Baker Institute for Animal Health, College of Veterinary
Medicine, Cornell University
Oral
Corticosteroid Treatment of Dogs Infected with Borrelia burgdorferi
Reinhard K. Straubinger, Alix F. Straubinger, Richard H. Jacobson,
Brian A. Summers James A. Baker Institute for Animal Health,
the NYS Diagnostic Laboratory, and Department of Pathology, College
of Veterinary Medicine, Cornell University, NY.
Corticosteroids
are powerful drugs often used to abrogate clinical signs of inflammation.
Therefore, administration of these drugs during Lyme disease
might be beneficial. However, they could be detrimental as well,
since corticosteroids impair the immune system. To investigate
the effects of corticosteroids on subclinical persistent B.
burgdorferi infection we treated persistently infected dogs
with oral prednisolone.
Four
specific-pathogen-free beagles were infected by tick challenge.
Dogs were maintained for 505 to 581 days after infection. During
this time, all dogs were monitored daily for clinical signs and
body temperature changes. Serum antibody titers against B.
burgdorferi were measured every two weeks, and skin punch
biopsy samples were obtained every month for culture. At day
413 after infection, two dogs (2 and 3) received oral treatment
with Prednisolone (2 mg/kg body weight) twice a day for 14 consecutive
days. The same prednisolone treatment regimen was administered
starting on day 566 to dogs 3 and 4 which was 15 days before
tissues were cultured for the presence of B. burgdorferi.
One
month after tick exposure, all four dogs showed infection as
documented by seroconversion and by spirochete-positive skin
biopsy cultures. Clinical signs of acute lameness developed between
50 and 169 days after infection. Dogs 1-3 showed two, three,
and one episode of lameness, respectively, while dog 4 did not
become lame. Onset of lameness was sudden and clinical signs
resolved usually after five days. Within 90 days after infection,
serum antibody titers reached maximal levels and remained unchanged
throughout the experiment. Besides weight gain, no adverse effects
were observed during the course of the corticosteroid treatment.
However, 5 and 8 days after the treatment had stopped, dog 2
and 3 developed severe polyarthritis, which resolved without
treatment after an additional seven days. At the end of the experiment,
25 tissues of each dog were cultured in BSK II medium. Dog 1,
which received no corticosteroids, had 14 positive tissues, while
dog 2 which had received prednisolone three months earlier had
only one positive tissue (fascia of the right hind leg). Dog
3 and 4, which received corticosteroid treatment shortly before
testing, had 10 and 19 tissues positive, respectively.
In
summary, dogs persistently infected with B. burgdorferi
show no adverse affects during oral corticosteroid treatment.
However, once the treatment was terminated, severe polyarthritis
occurred, probably due to reactivated persistent infection triggering
local joint inflammation and an enhanced immune response against
B. burgdorferi.
Ron
Schell, PhD
University of Wisconsin School of Medicine
Overview
of Testing in Lyme Disease
No
abstract submitted
Steven
Schutzer, MD
University of Medicine and Dentistry of New Jersey
Immunodetection
of Borrelia burgdorferi in vivo Expressed Antigens
Precise
immunodiagnosis of infectious diseases is based upon identification
of an immune response to unique antigens of the suspected agent.
Temporal issues such as recent or past infection require understanding
and application of the immune response. Rapidly multiplying bacterial
infections may provoke an early IgM response which may suggest
acute infection. Early expression of antigens unique to the organism
may also provoke an antibody response which may suggest acute
infection. Borrelia burgdorferi (Bb) infection which causes
Lyme disease has a behavior different than a fast growing bacterial
disease. The immune response is often delayed by weeks, well
beyond the optimal clinical utility of current immunologic tests.
However the spirochete produces several antigens that are unique
to Bb, permitting selective diagnosis of this organism.
In addition to the Osp proteins, more recently discovered ones
such as 22 kd, 35, and 37, are produced only in vivo infections
as opposed to the in vitro cultured organisms. The detection
of antibody bound to unique antigens in Bb specific immune
complexes is a marker for active disease as opposed to past infection.
This has important diagnostic and therapeutic uses.
Nick
S. Harris, PhD
IGeneX, Inc. Reference Laboratory
Antigen
Detection of Borrelia burgdorferi in Urine
This
is a review of the relationship of the bladder and the urine
as a unique area for the detection of B. burgdorferi.
Since 1986, there have been reports on the presence of B.
burgdorferi spirochetes or antigen in urine, or cultured
from the bladder, of mice, rabbits and dogs in experimentally
and naturally induced Lyme infection. A variety of detection
techniques have been used: 1) biopsy; 2) culture; 3) PCR; and
4) antigen-capture assays. In human systems, both PCR and antigen-capture
with monoclonal or polyclonal antibodies have been used.
While
the PCR technique seems to be more useful prior to antibiotics
or after antibiotics have stopped, in those patients with persistent
disease, the detection of antigen in urine by Antigen-Capture
may actually be enhanced during antibiotic treatment. There have
been studies, using nested PCR, showing the consistency of detecting
DNA antigen in urine, early in disease, during the time of the
EM. In another study, multiple primers sets have been used to
detect B. burgdorferi DNA in patients with chronic (persistent/recurrent)
Lyme disease. The LUAT (Lyme Urine Antigen Test), developed in
1990, utilizing a unique polyclonal antibody and a variant of
the fluorescent ELISA has detected antigen in patients with early
disease as well as those with persistent/recurrent disease.
Data
from the PCR and Antigen-Capture studies will be used to illustrate
the fact that the urine is an important tissue for the clinical
detection of laboratory markers for Lyme disease.
Paul
Duray, MD
National Institutes of Health, National Cancer Institute
New
System for Borrelia in vitro Cultivation
Paul H. Duray, MD, Steven Hatfill, MD, PhD, Adriana Marques,
MD, Leonid Margolis, PhD
Most
in vitro cultivation systems are unicell type cultures
involving one cell lineage type or another. The current technology
is limited concerning the feasibility of growing whole tissue
fragments with integral mesenchymal stroma, extracellular matrix,
blood vessels, and epithelial structures, although some investigators
have maintained thin tissue fragments for a limited time in matrigels.
NASA engineers at the Johnson Manned Spaceflight Center designed
and developed a fluid filled Bioreactor called a Rotating Wall
Vessel Bioreactor (RWV), a double-walled instrument which operates
under microgravity conditions. The instrument was originally
developed to conduct experiments in space on living cells and
tissue samples. Rotation and microgravity conditions afford the
efficient transport of cell nutrients and the withdrawal of toxic
metabolites, enabling the growth of integral stroma and parenchymal
tissues for extended periods of time. Basic construction is a
cylindrical growth chamber containing an inner co-rotating cylinder
with a gas exchange membrane. Viscous coupling causes the fluid
medium in the inner vessel to accelerate until the entire fluid
mass is rotating at an angular rate equal to that of the outer
wall, resulting in beads, cells or tissue chunks to remain suspended
in the fluid at a given rotational speed. We and others have
learned that mammalian and human tissue samples are maintained
viable for extended periods of time, permitting the feasibility
of conducting infectious disease and carcinogenic experiments
without resorting to laboratory animal experimentation. Examples
of human tissue that can be maintained viable in the RWV include
spleen, lymph node, tonsil, salivary, skeletal muscle, synovial,
lung, skin, and prostate tissue. For reasons unknown, we have
been unsuccessful with CNS and renal tissue. We recently cocultured
Borrelia burgdorferi with human tonsil, skin, and synovial
tissue obtained from routine surgery excisions, and showed that
the spirochetes preferentially invaded the rotating tissue samples,
and grow in numbers in the tissue that far exceed that seen in
such tissue samples under conditions of natural infection. This
system enables the maintenance of Borrelial spirochetes
in mammalian and human tissue samples, ex vivo, and may be used
as an adjunct to isolate miroorganisms from clinically derived
biopsy samples of erythema migrans, joint and soft tissue, and
other sites.
Charles
Pavia, PhD
Associate Professor, New York Medical College-WCMC
Department of Medicine, Division of Infectious Diseases
Improved
Culturing and Sensitivity to Antibiotics of Bb Isolated
from patients with Early Lyme
Despite
dissemination during early Lyme disease, the yield of blood cultures
for detecting B. burgdorferi is often 5% or less. The
volume of blood cultured influences the yield in other bacteria
infections. Whether increasing the volume of blood or serum inoculated
into media would improve the yield of blood cultures in early
Lyme disease is not known. In our initial studies, three 3cc
aliquots of whole blood or serum were inoculated into BSK media
from 31 patients presenting with erythema migrans. Eight (25.8%)
of the 31 patients had a positive whole blood or serum culture,
including 3 of 6 (50%) with multiple lesions compared to 5 of
25 (20%) with a single lesion (P=).16). Whole blood was culture-positive
for 3 of 30 (10%) evaluable patients compared to serum which
was positive for 6 of 31 (19.4%) patients. In subsequent studies,
six 3cc aliquots of serum were inoculated into BSK media from
26 untreated patients with EM. Seven (27%) patients were culture-positive
including 2 of 7 (28.5%) with multiple lesions and 5 of 19 (26%)
with a single lesion. We conclude that B. burgdorferi
can be recovered from peripheral blood in 25% of patients presenting
with the EM rash if sufficient volume is inoculated into culture
media. Some of these new patient isolates were tested for their
sensitivity in vitro to selected antibiotics. When compared
to standard laboratory-adapted strains, such as B31 and CA287,
these new isolates were highly susceptible to antibiotics at
concentrations equal to or less than 0.5 ug/ml.
Julie
Rawlings, MPH
Texas Department of Health, IDEAS
Tick-borne
Disorders
The
risk for acquiring tick-borne diseases increases as the weather
gets warmer and ticks begin looking for blood meals. Health care
providers should be cognizant of the many tick-borne diseases
that occur in the United States, including babesiosis, caused
by protozoa of the genus Babesia; Colorado tick fever,
caused by a double stranded RNA virus; Lyme disease and tick-borne
relapsing fever, caused by Borrelia spirochetes; and Rocky
Mountain spotted fever, ehrlichiosis, and tularemia caused by
small gram-negative coccobacilli. Vectors for the agents of these
diseases include the hard ticks Amblyomma americanum, Dermacentor
andersoni, Dermacentor variabilis, Ixodes pacificus, and
Ixodes scapularis and soft ticks Ornithodoros hermsi
and Ornithodoros turicata. Signs and symptoms for the
various tick-borne diseases may be similar; these, as well as
their transmission cycles, laboratory diagnosis, and treatment
will be discussed.
William
T. Golde, PhD
Division of Allergy, Dept. of Medicine, SUNY at Stony Brook School
of Medicine
Coinfections
in Patients on Long Island
Ixodid ticks
are known to be the vectors of Borrelia burgdorferi and
the piroplasm Babesia microti. In addition, these tick
species are also believed to be the vector of the agent of human
granulocytic ehrlichiosis (HGE). We conducted a prospective study
in 1997 to analyze patients presenting with erythema migrans,
and therefore Borrelia burgdorferi infection, for co-infection
with two other pathogens transmitted by the vector of Lyme disease
on Long Island, Ixodes scapularis. In the course of our
study, we enrolled 18 patients from which we obtained serum and
whole blood samples and 16 of which submitted to a skin punch
biopsy. Serum samples were assayed for antibody to all three
pathogens, whole blood was tested for the presence of these infectious
agents by PCR and direct culture, and biopsies were cultured
as well as analyzed by PCR. Results indicate that the co-infection
rate for B. burgdorferi and the agent of HGE is very high
where as the rate of infection with B. microti is low.
The continued development and improvement of detection methods
and recruitment of a larger patient population are ongoing in
order to confirm these preliminary findings.
Richard
C. Tilton, PhD
BBI Clinical Laboratories, Inc.
Confirmatory
Testing for Non-Lyme Tick Borne Diseases
A
variety of tests are available for laboratory diagnosis of Babesia
and both types of Ehrlichia infections. The mainstay of
antibody screening for both Babesia and Ehrlichia
is an indirect fluorescent antibody test (IFA). IFA slides have
become commercially available but if the situation is analagous
to Lyme disease testing, a confirmatory test should be performed.
Results
are presented on a variety of IFA test for Babesia antibody
as well as a confirmatory Western Blot for IgG and IgM antibodies
to Babesia.
Over
500 Patients were tested for antibodies to both ehrlichial species
during the summer of 1996. All reactive IFAs were reflexed to
a Western blot. Results indicate that a Western Blot confirmatory
test is indicated for HGE but, at this time, not for HME.
Paul
Duray, MD
National Institutes of Health, National Cancer Institute
Host
And Mammalian Histopathology In Borreliosis
Basic
histopathologic alterations in human target tissues in LD involve,
with some geographic differences, the presence of T lymphocytes
with subsets, abundant plasma cells and precursors, macrophages,
dendritic antigen-presenting cells, variable mast cells, synovia,
chronic skin and soft tissue sites, particularly striated muscle,
and infrequently, peripheral and in the acute stages, some neutrophiles.
Over time, the immunopathologic infiltrates aggregate in joint
nerves and interstitial lymphohistiocytic inflammation. Myocarditis
is thought to be transient, but when present is comprised of
transmural interstitial lymphohistiocytic inflammation. There
are some geographic differences: lymphocytic nodular aggregates
of skin and muscle are found in European cases, with proliferative
synovitis resembling rheumatoid arthritis more common to U.S.
Acrodermatitis
chronica atrophicans with a wide spectrum of histopathologic
changes is nearly exclusive of U.S. cases, more commonly seen
in Europe. Unusual cases of CNS involvement, splenitis, progressive
uveitis, eosinophilic fasciitis, tenosynovitis, and morpheaform
skin lesions have been associated with infection, but too infrequent
to be considered components of human borreliosis at this stage
of current knowledge.
Domestic
pet infections mimic human lesions regards autonomic gangliitis,
myocarditis, and synovitis. Rodents and lagomorphs in boreal
sites have infrequent inflammatory foci in livers, but no synovial,
CNS, or soft tissue alterations that are consistently seen. White
tailed deer examined from Westchester County, NY, had lymphoid
hyperplasia of spleen with stainable spirochetes, and hepatic
nodular lymphocytic infiltrates. These deer harbor stainable
spirochetes in the ocular vitreous. Southern Wisconsin deer have
similar findings. Regards induced infection in laboratory animals,
Lewis rats incur skin and joint inflammation, myocarditis and
soft tissue inflammation in NIH-3 mice, and erythema migrans
in lagomorphs.
This
experience has been accumulated from human samples where there
had not been prior antimicrobial treatment before tissue sampling.
In general, spirochete searches microscopically, are unsuccessful
in patients having had prior antibiotic therapy.
Anthony
Lionetti, MD
Lyme Disease Diagnostic Center
Early
Localized Lyme Disease
This
is a general presentation of currently accepted medical practice
of the clinical manifestations, laboratory evaluation, diagnosis
and management of early localized human Lyme disease.
Dennis
Parenti, MD
SmithKline Beecham Pharmaceuticals & Biologicals
Erythema
Migrans and Sero-epidemiologic Findings from SmithKline Beecham's
Lyme Vaccine Trial
D. Parenti, D. Krause, SmithKline Beecham Biologicals and the
Lyme Disease Vaccine Study Group, Collegeville PA
Erythema
migrans (EM) is the most common presenting symptom of Lyme disease.
Textbooks and early descriptions state that it accounts for 50-80%
of the cases, although recently some authors claim that EM may
be the presenting symptom in greater than 90% of cases. The EM
rash has been classically described as a "bulls-eye"
rash with central clearing occurring at the site of the tick
bite. There are only a few authors who have described the morphologic
findings, including atypical appearances, from large series of
EM lesions.
SmithKline
Beecham has recently concluded a double-blind, placebo-controlled
trial of it's candidate vaccine, LYMErix (recombinant Lipoprotein
OspA with adjuvant) for Lyme disease. In this study of over 10,900
volunteers, subjects who developed a rash were evaluated with
photographs, skin biopsies for culture and PCR as well as acute
and convalescent sera for Western Blot testing. Serological results
were compared to baseline sera drawn at study start for evidence
of seroconversion.
There
were 142 laboratory confirmed cases of EM diagnosed during the
study. Examples of typical EM as well as the most common and
atypical appearances will be displayed. Data regarding the incidence
of positive cultures and PCR will be discussed. In addition,
epidemiologic data regarding baseline serologic findings will
be presented.
Kenneth
Liegner, MD, PC
Internal & Critical Care Medicine
Lyme
Borreliosis and Related Disorders
Dissemination:
- Movement
of the Lyme organism from the site of entry into the host to
remote sites within the body
- May
be hematogenous, via lymphatics, or by direct invasion along
tissue planes
- Plasminogen
activation may facilitate direct tissue plane invasion
- Minimum
tissue to dissemination unclear but may be very early possibly
within days of occurrence of erythema migrans
- Dissemination
early following infection may set the stage for chronicity with
constitutional and multi-system symptomatology and late organ
system involvement
- Intracellularity
has been demonstrated in vitro but not in human disease. If it
occurs, this may help explain both chronicity and refractorisness
to antibiotic therapy.
Diagnosis
of Disseminated Lyme disease: Clinical Features
- CDC
has always emphasized Lyme disease as a clinical diagnosis; lab
data supportive only
- Spectrum
of illness: expanding, and limits of clinical manifestations
presently unknown: need for open mindedness in considering what
may or may not be "Lyme disease"
- Correct
diagnosis requires good understanding of Lyme disease and a good
fundamental knowledge of basic medical knowledge is necessary
considering the potentially broad differential diagnosis of Lyme
disease with its many manifestations
- Detailed
geographic history is crucial; consider that most human beings
in the course of their lives have epidemiologic exposure risk
for ticks: summer vacations/summer camp; visits to shore or mountain
areas; residence in or visits to Lyme endemic areas vocational
& avocational pursuits in tick infested areas
- Bear
in mind a low inoculum of spirochetes may take months or years
to attain a body burden of infection sufficient to case clinically
evident symptomatology; we don't know the minimum infectious
dose of borreliae sufficient to cause infection and clinical
disease. Time from first manifest signs of illness to diagnosis
also may entail many years during which direct and immune-mediated
injury can occur.
- Constitutional
complaints w/multi-system symptomatology although non-specific,
actually rather characteristic. A tip off is these occur in previously
essentially healthy individual: fatigue; arthralgia & myalgia,
often migratory; cervicalgia; recurring low grade fever; night
or day sweats; recurring sore throat & swollen glands; paresthesisa;
sleep disturbance; enthesitis; panic attacks; anxiety; cognitive
difficulties; mood disturbance; a "sick" feeling, malaise
; often cyclic with 4-6 week cycles
- Often
look "well", may exhibit few physical findings. Sometimes
a veritable "blizzard" of symptoms that threaten to
overwhelm the physician, as it has overwhelmed that patient.
Psychosomatic Yet multiple individuals proving to have disseminated
Lyme disease report nearly identical phenomena, almost carbon
copy.
- Occasionally
isolated symptom or organ involvement and thus lacking the multi-system
"flavor"
Organ
systems:
- Central
nervous system: Brain, brain stem, and spinal cord: meningitis;
meningoencephalitis; meningoencephalomyelitis; myelopathies;
transverse myelitis; hemiparesis; paraparesis; spastic para-
and tetraparesis; motor neuron disease; extrapyramidal syndromes/choreiform
syndromes; locked-in" state; coma; progressive leukoencephalopathies;
multiple sclerosis-like syndromes; seizure disorders; cerebral
atrophy; organic brain syndromes/dementia; encephalopathy; neuropsychiatric
syndromes: psychoses; OCD; depression: mania; bipolar disorders;
other psych. syndromes. Cranial nerve palsies involving any CN
multiple CNS may be involved; radiculoneuritides; sciatica-like
syndromes; neurogenic bladder
- Peripheral
nervous system: peripheral neuropathies; motor/sensory/plexopathies;
paresthesias/dysesthesia
- Autonomic
nervous system dysfunction: cardiogenic syncope and vasodepressor
syncope; abdominal bloating and abnormal peristalsis
- Auditory
& vestibular apparatus: tinnitus; disturbances of balance;
vertigo; hyperacusis; hearing loss
- Ocular:
all levels, all structures of eye may be involved; conjunctivitis;
keratitis; uveitis; optic neuritis; retinitis/retinal vasculitis;
cataract formation; retrobulbar myositis; optic cortex cerebritis
- Musculoskeletal:
arthralgia; arthritis/synmovitis; myositis/myopathy; painful
myalgia/fibromyalgia-like syndrome; muscle fasciculation; fasciitis;
enthesitis
- Genitourinary:
neurogenic bladder; interstitial cystitis"; renal damage/glomerulonephritis?
(Reported in dogs, so far cases in humans have not been reported)
- Endocrine:
thyroiditis?; Effects on libido; central hypothalamic ?; orchitis;
cyclic flare of symptoms temporally related to menstrual cycle
in women.
- Cardiac:
dysrhythmias; heart block; various types of extra-systoles; autonomic
dysfunction: cardiogenic syncope, vasodepressor syncope; cardiomyopathy;
congestive heart failure; myopericarditis
- Gastrointestinal:
bloating; GERD?; Irritable bowel/colitic presentations?; Myoenteric
autonomic dysfunction?; Abdominal pain/cramping esp. in children;
Lyme hepatitis; Lyme enterocolitis?
Differential
diagnosis:
- Multi-system
involvement - not too many things do this e.g. joint and neurologic
involvement: CTDs; syphilis; sarcoidosis; chronic viral infections
(hepatitis/HIV/CMV/parvovirus)'; TB; brucellosis; relapsing fever;
parasitic disease
- Mimicking
other multi-system and autoimmune diatheses:
RA-like w/ ^ RF; often RF decrease with antibiotic RX
Lupus-like w/ ^ ANA, Anti-DS DNA AB; ^ c1Q I.C.s etc. seositis/pericarditis/thyroiditis/
etc. markers may diminish with ABx Rx
Mimicking the Chronic Fatigue Syndrome
Mimicking Idiopathic fibromyalgia
Disseminated
Lyme disease: Laboratory Diagnosis
- Lyme
ELISA and Western blot: Over-emphasis on "false positive"
ELISAs; Is Late Lyme/disseminated disease almost invariably seropositive?
- Seronegativity:
Real or Bogus? Many of the culture proven cases of Lyme disease
in the world literature occur with seronegative patients. Study
in our practice specializing in Lyme disease in last quarter
of 1996: 16% of patients had positive Elisas and positive Western
blots whereas 21% of patients had dead negative Elisas and fully
diagnostic IgG or IgM, Western blots; many others had suspicious
Wbs having less than 5/10 "CDC-specific" bands (Kochevar
& Liegner). Always request reporting of ALL bands on a Lyme Western
blot.
Useful
laboratory tests:
- ESR;
C-Reactive Protein; Lyme Elisa and Western blot to two GOOD laboratories;
always ask that ALL bands present be reported!; CBC w. Diff;
Chemistry profile; ANA; FTA-ABS, if +, MHA-TP; TFTs w TSH; Angiotensin-1-converting
enzyme; Anticardiolipin antibodies; Quantitative immunoglobulins
(frequent polyclonal IgM elevation, occas IgG ^)
- Histologic
demonstration by silver staining in biopsies or tissues removed
at surgery; role for electron microscopy - (must be fixed in
glutaraldehyde)
- Research
Assays: role being explored; PCR in any body fluid; tissue PCR;
LUAT (Lyme urine antigen test); Gundersen (borrelial immobilization)
test; Lyme-specific immune complexes - Coyle/Schutzer Elisa-capture
Osp A and Osp B antigen detection CSF
- Direct
culture BSK-II (Barbour-Stoenner-Kelly media)
- CSF
examination; Paired Lyme Elisas and paired Western blot in serum
and CSF; Multiple sclerosis panel,; Cytology; Cell count & differential,
glucose & protein; VDRL; CSF viral culture and if appropriate,
viral titers, PCR for detection of Bb-specific DNA; If available,
OspA and/or OspB antigen detection ample CSF so excess can be
stored frozen (in a non-cycling freezer) indefinitely for possible
future study.
Other
useful adjunctive diagnostic studies: MRI; Brain SPECT; Detailed
neuropsychological testing; NCS/EMGs
Disseminated
Lyme Disease: Approach to Treatment
- Cookbook
approach inappropriate
- Duration
of treatment should be based upon clinical response
- In
most instances trial of oral RX is appropriate before resorting
to intravenous antibiotic therapy. Since many patients with disseminated
Lyme disease can be adequately treated with oral therapy.
- Careful
periodic assessment of the patient by the physician is essential.
- Several
months of treatment may be necessary to assess response to RX
- Full
discussion of risk/benefits of treatment
- Careful
periodic monitoring of the patient is necessary to detect any
adverse consequences of antibiotic therapy CBC, chem, U/A usually
monthly to quarterly depending on agent
- Attention
to gut hygiene with acidophilus 2 hr. following oral antibiotic
dosage
- Anticipate
and try to deal early with any complications of Rx (e.g. C. difficile
etc., yeast overgrowth)
Oral
antibiotic therapy: monotherapy:
tetracycline class
tetracycline (TCN) 500 T.I.D.
doxycycline (DCN) 100-200 mg Q 12 hr.
Minocycline (MNCN) 50-100 mg Q 12 hr.
amoxicillin
.5-2 rams TID with or without probenicid
cefuroxime (Ceftin) 500-1000 mg Q 12 hr
azalide class
clarithromycin (Biaxin) 500-1000 mg. Q 12 hr azithromycin
(Zithromax) 250-500 mg. Q 12 hr
Combined
oral antibiotic therapy:
amoxicillin + TCN, DCN, or MNCN
amoxicillin + an azalide
cefuroxime + a TCN class or azalide class agent
Intravenous
Rx: monotherapy
ceftriaxone (Rocephin) 2 QD
cefotaxime (Claforan) 6 grams/day
imipenem/cilastatin (Primaxin) 250-1000 mg. Q 8 hr
doxycycline 100-200 mg. IV Q 12 hr
vancomycin 500-1000mg. IV Q 12 hr
azithromycin (Zithromax) 500mg IV QD
amipicillin 1-2 grams IV Q 6 hr
penicillin G 12-20 million units/day
Role of Empiric
Diagnostic and Therapeutic Trial:
Important and legitimate role for empiric trial of treatment
in appropriate clinical setting after detailed and thorough evaluation;
may be appropriate even in the absence of any laboratory proof
of diagnosis. Onus is on physician to have carefully excluded
other identifiable and treatable conditions.
Monitoring
of Response to Rx:
Meticulous clinical assessment; Patient subjective report; Physical
examination; Serial neuropsychological testing; Serial Western
blots; Serial direct antigen detection methods, particularly
at time of clinical relapse; Serial MRIs, SPECTs, NCS/EMGs; Serial
CSF examinations if perturbed parameters present initially.
Robert Lesser,
MD
Yale University
Eye Findings
in Lyme Disease
Conjunctivitis;
Keratitis; Uveitis; Optic Neuritis; Optic Atrophy; Pupillary
Abnormalities.
Brian A. Fallon,
MD, MPH, MEd
The NYS Psychiatric Institute
Neuropsychiatric
Aspects of Lyme Disease in Children and Adolescents
Brian A. Fallon, MD, Felice Tager, Marian Rissenberg
Although Lyme
disease in adults is known to be associated with cognitive and
psychiatric problems, little research has been conducted on the
neuropsychiatric aspects of Lyme disease in children and adolescents,
even though the infection rates among children are high. Previous
reports on children have found little evidence of neuropsychiatric
sequelae, however these reports were conducted largely among
children with recently diagnosed Lyme disease who presented with
an erythema migrans rash, thereby precluding generalizations
to the sample of young patients with chronic Lyme disease. In
this talk, recent research results from our Lyme Disease Research
Center and other sites will be reviewed.
In one ongoing
study under the direction of Felice Tager, 20 children with Western-blot
positive chronic Lyme disease were compared to 15 healthy control
children age 9-17 on a battery of neuropsychological tests. The
Lyme patients had significantly higher rates of psychopathology
including feelings of incompetence, social withdrawal. anxiety/depression,
trouble thinking, attention problems, and aggressive behavior.
Cognitively, the Lyme patients had significantly higher rates
of deficits in visual scanning and tracking, in verbal memory,
and in the freedom from distractibility index of the WISC-III
(a measure of attentional problems). On a continuous performance
test, the Lyme children also had significantly worse performance.
These results,
while preliminary, demonstrate that children and adolescents
with chronic Lyme disease experience significant problems psychiatrically
and cognitively. The impact of these problems on the child, family,
and school will be addressed.
Anthony Lionetti,
MD
Lyme Disease Diagnostic Center
Clinical Characterization
and Serological Data on 200 Patients PCR Positive For Lyme Disease
Objective:
There have been few studies that have studied the serological
response and clinical characterization in patients who have been
proven to be infected with Borrelia burgdorferi by a direct
detection technology. A retrospective analysis is presented of
the serological and clinical data of 200 consecutive patients
who tested positive for infection by Borrelia burgdorferi
sensu lato utilizing a highly specific nested PCR of blood
and/or urine from 1994-1998.
Methods: A
retrospective chart review was performed, extracting the data
from the clinical notes and laboratory testing. Serological testing
and PCR testing was performed by a single laboratory recognized
for accuracy and reliability in Lyme disease testing by published
peer reviewed laboratory proficiency testing. Other items registered
included patient age, sex, residence, tick bite history, and
duration of disease.
Results: In
this group of 200 consecutive patients primarily confirmed for
infection with Borrelia burdorferi sensu lato by PCR,
there was poor confirmation by IgG/IgM immunoblotting. In IgG
there were 68% non reactive, 31% equivocal, and <1% reactive.
In IgM there were 64% non reactive, 22% equivocal, and 14% reactive.
Conclusions:
In this group of patients there was poor correlation between
direct molecular genetic proof of infection with Borrelia
burgdorferi sensu lato and Lyme IgG/IgM immunoblotting. Explanations
for this lie in issues such as:
- Duration
of infection
- The effects
of previous treatment on the serological response
- Antigen-antibody
immune complexes which may prevent the availability of free antibody
available for detection
- Analysis
of interpretative criteria for Lyme immunoblotting with evaluation
of other algorithms for improving the sensitivity of this test.
Edwin Masters,
MD
Regional Primary Care
Clinical Borreliosis
in Missouri
Examples of
clinical erythema migrans in Missouri are presented along with
other clinical presentations and examples of sequelae. Etiological
and epidemiological theories are presented and discussed. This
includes the hypothesis that just as there exists a clinical
triad of Ixodes scapularis vectored borreliosis (Lyme
disease), ehrlichiosis (HGE), and babesiosis microti in
the Northeast, there may also exist in the South, including Missouri,
a similar clinical triad vectored by lone star (Ambylomma
americanum) ticks. Patients with signs and symptoms explained
only by a borreliosis following lone star tick bites are presented.
Babesia MO 1 has been identified in a Missouri patient
and although vector studies have not been done, other Babesia
are known to be carried by Amblyomma ticks.
Ehrlichiosis
(HME) is known to be carried by lone star ticks in the South.
Parallel evolutionary path in these two tick lines might explain
what clinicians around the United States are seeing. Each of
the three illnesses might have northern variants associated with
Ixodes scapularis ticks and southern variants associated
with lone star ticks. The clinical disease variants appear clinically
similar, but have testing, microbiological, and culturing differences.
Ehrlichiosis represents the prototype for this theory. Recent
evidence of tick to tick (even different species) transmission
of Borrelia burgdorferi while feeding on hosts and the
isolation of B. burgdorferi sensu lato from a lone star
tick feeding on a rabbit at one of my Missouri erythema migrans
patient's farm are both consistent with this theory. More research
is needed.
Sam Donta,
MD
Professor of Medicine, Boston University Medical Center
Chronic Lyme
Disease
Patients who
develop persisting symptoms after an initial episode of Lyme
disease are often referred to as having chronic Lyme disease
or post-Lyme syndrome. There are numerous other patients who
never recalled having a tick bite or a rash who also develop
what appears to be the same clinical disease. Often, depending
on the results of serologic testing, these patients are given
the diagnosis of chronic fatigue syndrome or fibromyalgia. The
etiology and pathophysiology of these multisymptom disorders
remain to be delineated. The major symptoms in all of these Lyme-like"
diseases consist of fatigue, musculoskeletal pains, and neurocognitive
dysfunction, and it is not readily possible to distinguish these
diseases on clinical grounds alone. In the case of chronic Lyme
disease, the organism or its DNA can be detected, albeit rarely,
suggesting that there is a persistent, intracellular infection.
The response to certain antibiotics also supports the idea that
this is a persisting infection. Additional clinical and experimental
evidence suggests that the reservoir is the nervous system, perhaps
in the sensory ganglia, as well as in the temporal and frontal
lobes of the brain The possibility that there are borrelial toxins
that interfere with normal neurochemical function is an idea
that is being further investigated.
Adriana R
Marques, MD
Head, Clinical Studies Unit, Laboratory of Clinical Investigation
National Institute of Allergy and Infectious Disease, National
Institutes of Health
Update on
the NIH Intramural Chronic Lyme Disease Study
Lyme disease
has become a highly controversial illness. The issue that has
probably generated the most controversy today is the mechanism
underlying persistent signs and symptoms of disease, despite
the administration of what is currently considered to be adequate
antibiotic therapy. Determining whether chronic Lyme disease
is caused by persistent infection or is a post-infectious disorder
is a fundamental issue. Finding the answer to this question for
any individual patient will have an important bearing on his
or her treatment, as our approach to the disease would be different
depending on the underlying mechanism.
To try to
answer some of these questions, we developed a new study in collaboration
with scientists in National Institute of Allergy and Infectious
Disease, in the National Institute of Neurological Disorders
and Stroke (NINDS), in the National Institute on Deafness and
Other Communication Disorders (NIDCD), in the National Institute
of Mental Health (NIMH) and with leading Lyme disease specialists
at outside institutions.
The objectives
of this study include evaluation of diagnostic laboratory abnormalities
and their correlation with the various syndromes; assessment
of the extension of infection with B. burgdorferi and
its consequences to patients; and the study of the role of immune-mediated
and other pathogenic mechanisms in injury to the nervous system,
including spirochete interactions with the immune system, auto-antibodies,
cytokines, cellular immune responses, and immune complexes.
The study
is now open for accrual and 29 patients have been enrolled to
date. At this point, it is too early to draw conclusions from
the analysis of the results of the multiple and extensive testing
done in the enrolled patients, but is our hope that these initial
studies involving very selected patients will provide new information
about chronic Lyme disease, and suggest additional avenues for
patient care and research.
Sam Donta,
MD
Professor of Medicine, Boston University Medical Center
Macrolide
Antibiotic Therapy of Chronic Lyme Disease
Evidence is
accumulating that patients with chronic Lyme disease respond
to certain antibiotic treatments. The organism responsible for
the infection is sensitive to several antibiotics in vitro, but
their clinical efficacy remains to be further evaluated. Further
questions exist regarding the location of the organisms and their
state of metabolism or reproduction. A leading hypothesis is
that these organisms are in intracellular compartments, and our
previous results with tetracycline support that hypothesis. In
contrast, the efficacy of macrolide antibiotics, which have excellent
in vitro activity against B. burgdorferi and excellent
intracellular penetration, has been unreliable.
Because macrolide
antibiotic activity is very restricted at an acid pH, it was
postulated that the borrelia may reside in acidic intracellular
vesicles and that the addition of a lysosomotropic agent would
improve the clinical activity of macrolides in Lyme disease.
Reported here are the results of studies of 235 patients with
a clinical diagnosis of chronic Lyme disease using a combination
of the lysosomotropic agent hydroxychloroquine and a macrolide
antibiotic. All patients had a clinical picture compatible with
chronic Lyme disease. Less than half recalled a tick bite or
rash. The EIA was positive in only 27% of patients, and the Western
Blot positive in 76%. Brain SPECT scans were positive in 73%
of patients. Overall, 80% of patients had significant improvement
or were cured; there were no obvious differences among the three
macrolide antibiotics used. Compared to patients ill for less
than 3 years, the onset of improvement was slower, and the failure
rate higher in patients who had been ill for longer time periods.
The encouraging results of these studies provide the basis for
additional treatment options and controlled studies in patients
with Lyme disease.
Irwin T. Vanderhoof,
PhD
NYU Stern School of Business
Lyme Disease
- Cost to Society A Catalogue of Symptoms
In the last
year there has been discussion of the costs justification for
early treatment of Lyme disease. Several arguments have been
presented based only on the relative costs of early treatment
for a large group vs. a course of antibiotics for those cases
confirmed by tests or by clinical diagnosis. This short course
of antibiotics is presumed to be effective in all cases.
Such calculations
can only seem justified if the calculation ignores the extreme
problems of those cases which do not respond to a short course
of treatment administered well after infection has taken place.
This presentation
is based upon data provided by the joint data base of the Lyme
disease foundation and the Society of Actuaries. In "Lyme
Disease: The Cost to Society (Contingencies, Jan.-Feb.,
1993, pp. 42-48, Karen Vanderhoof-Forschner and Irwin T. Vanderhoof)
information on these costs were detailed and an estimate of the
total cost of Lyme disease to society at &1 Billion per year
was developed. The current presentation is based upon the somewhat
larger data base now available. It confirms and supports the
earlier analysis and confirms the relation between these costs
and the length of delay from time to infection and treatment.
The costs for the 771 diagnosed cases in the data base totaled
$52,000,000. This amount would justify a large number of early
treatments. In addition a clear relationship is demonstrated
between the dollar costs of the disease, treatment and lost income,
and the delay in treatment of these cases.
Data was available
in the questionnaire concerning the outcome of pregnancies. This
data had not been previously analyzed. 55 live births were reported
by women diagnosed with Lyme disease. Also 19 miscarriages and
7 neonatal deaths were reported by this group. According to the
Statistical Abstracts of the United States in 1992 7.4 fetal
and 5.4 neonatal deaths were reported for each 1,000 live births.
We would then have expected a total of one such early death for
the 55 live births in our data base. The difference is statistically
convincing. Lyme disease seems to be a significant risk factor
for pregnancy. The separate pregnancy register of the Lyme disease
foundation provides a similar result. Out of 732 entries there
were 148 reported as abnormal births.
Because of
the importance of early diagnosis an analysis was made of the
symptoms in an attempt to establish their frequency for this
group of intransigent cases. In an attempt to establish whether
or not certain groups have the same sets of symptoms; the Hotelling
T2 test was applied. This test makes comparisons using all the
factors at once. If the symptoms constitute 55 weak indications
of the disease then taken together they might become a stronger
indicator. These tests showed that the symptoms for men and women
were different, that those who had a tick bite and exhibited
a rash and had positive tests had somewhat different symptoms
from other diagnosed cases, and that cases from Minn. and Wis.
had a somewhat different pattern from those in the rest of the
country.
On the other
hand cases reported from cooperating physicians were not different
than all other cases. In addition, cases that responded to a
follow up questionnaire on joint swelling did not differ from
those not responding. This Hotelling test seems not to have been
frequently used in medical studies. It has the advantage of being
able to confirm that the results of one group can be justified
in applying to differently selected group.
Finally this
study details the pattern of symptoms that would be expected
to mentioned to the attending physician. In addition to the significant
symptoms the data indicate that over 4 of 7 designated systems
of the body, on average, would be effected.
Denise M.
Foley, PhD
Assistant Professor, Chapman University, Orange, CA
Potential
Limitations of the OspA Vaccine for Humans Based Upon Experimental
Studies in Animals
Denise M. Foley, PhD, Chapman University, Orange, CA and James
N. Miller, PhD, UCLA, Los Angeles, CA
The apparent
success achieved in human trials in the United States with recombinant
OspA lipoprotein vaccine has now been reported. However, several
studies in animals, including those emanating from our laboratory,
have revealed potential limitations that may be associated with
its use. In this presentation, we will review published animal
studies which demonstrate several of these limitations. Issues
to be discussed include 1) the relationship of OspA heterogeneity
among North American isolates and lack of expression in vertebrates
as it relates to potential infection after vaccination, 2) potential
low level infection (latency) and/or an altered disease state
following exposure leading to misdiagnosis and subsequent disseminated
disease among some vaccines following exposure.
Dennis Parenti,
MD
SmithKline Beecham Pharmaceuticals & Biologicals
The Safety
and Protective Efficacy of an Adjuvanted Lyme Disease Vaccine
D.L. Parenti, C. Buscarino and D.S. Krause. SmithKline Beecham
(SB) Pharmaceuticals & Biologicals, Collegeville, PA, and the
Lyme Disease Vaccine Study Group
A safe and
efficacious vaccine against Lyme Disease (LD) is needed due to
the progressive increasing incidence and geographical spread
as well as the inadequacy of personal protection measures. A
vaccine must also protect against asymptomatic infection as well
as clinical disease. We conducted a double-blind, phase III vaccine
efficacy (VE) study for the prevention of LD, in 31 centers,
in LD endemic areas.
Approximately
11,000 volunteers (ages 15-70) received LYMErix™ (30 mcg
lipoprotein OspA, adjuvanted vaccine), or placebo (1:1) on a
0,1,12 month schedule. Subjects were followed for 2 LD seasons.
Sera were drawn on all subjects at baseline, months 12 and 20
for Western blot testing to detect asymptomatic infection. Subjects
with suspected LD provided biopsy specimens for culture and PCR,
acute and convalescent sera and other appropriate lab specimens
to detect infection.
After 3 doses,
there were 13 laboratory confirmed cases of LD in the vaccinees
[Attack rate (AR) = 0.27] and 61 in the placebo group (AR = 1.28,
P: 0.001), yielding a vaccine efficacy (VE) of 79% (95% CI: 61-88).
Partial protection was already achieved after 2 vaccine doses
(VE 50%; 95% CI: 14-70; P = 0.01). VE was also high for asymptomatic
infection (83% after 2 doses, P = 0.008; and 100% after 3 doses;
P < 0.001). In vaccinees between 15-65 years, following 3
doses, the protective efficacy of the vaccine against laboratory-confirmed
B. burgdorferi and asymptomatic infection was 90% (95%
CI: 78-95; P = 0.001). Solicited local and general reactions
were common, but most were considered "mild" to "moderate"
by the subjects and were limited in duration. There were no unusual
pattern(s) of adverse events.
SB's LYMEri™
has an acceptable reactogenicity profile and is efficacious for
prevention of laboratory confirmed clinical LD as well as asymptomatic
infection.
Bob Huebner,
PhD
Pasteur Merieux Connaught
Strategies
for a Vaccine Against European Borrelia
Lyme disease
is the most prevalent tick-borne disease in the US and an important
tick-borne disease in Europe. The completion of successful clinical
trials with an OspA-based formulation in the US begs the question
as to how this success could be translated to a European Lyme
disease vaccine. Several factors must be critically considered
when formulating strategies for development of a European Lyme
vaccine. In Europe, Borrelia burgdorferi, the Borrelia
associated with Lyme disease in the US, and two additional genospecies
of Borrelia, Borrelia afzelii and Borrelia garinii,
cause Lyme disease. The symptoms associated with Borrelia
garinii or Borrelia afzelii infection suggest case
definitions used for clinical trials in the US will need to be
revised. Reports from European investigators describe Borrelia
isolates that either poorly express or don't express OspA, the
antigen used in US formulations. European isolates of Borrelia
show more variation in their OspA genes and those of other vaccine
candidates. The impact of these factors on the development of
a European Lyme vaccine strategy will be discussed.
David R. Cassatt,
PhD
MedImmune, Inc
Borrelia
burgdorferi
Decorin-Binding Protein A (DbpA) as a Second Generation Lyme
Disease Vaccine Candidate
David R. Cassatt, Nita K. Patel, William C. Roberts, Nancy D.
Ulbrandt and Mark S. Hanson. MedImmune, Inc.
The binding
of Borrelia burgdorferi to the collagen-associated extracellular
matrix proteoglycan decorin has been found to be mediated by
two lipoproteins, decorin-binding proteins A+B (DbpA, DbpB).
In contrast with OspA, antibodies to these proteins can be found
in chronically infected mice inoculated with low doses of Borrelia.
As reported in Infection and Immunity (Vol. 66, No. 5,
in press) we have found that active immunization with one of
these proteins, DbpA, can completely protect mice against homologous
challenge and partially protect mice against heterologous challenge
with Borrelia. Anti-DbpA serum had cross-strain borreliacidal
activity in vitro and in vivo.
We examined
the post-infection potency of anti-DbpA serum to determine whether
we could prevent infection of mice after the Borrelia were adapted
to the host environment and found that passive administration
of anti-DbpA, but not anti-OspA, sera could clear Borrelia up
to four days after infection, further suggesting that DbpA, but
not OspA, was expressed in host-adapted spirochetes. To obtain
direct evidence of in vivo DbpA expression, we have isolated
Borrelia from blood of infected mice and have performed immunofluorescence
and antibody growth inhibition assays on these in vivo- adapted
Borrelia. We report that antiserum raised against recombinant
DbpA bound the isolated Borrelia and that Borrelia incubated
with this antiserum was unable to subsequently form colonies
in soft agar plate culture. Antiserum raised against OspA did
not bind these isolated Borrelia nor did it inhibit colony formation
after incubation. Furthermore, immunization of mice with DbpA,
but not OspA, prevented infection from blood-borne Borrelia.
These studies demonstrate the possibility of targeting in vivo
expressed Borrelial antigens such as DbpA.
